Protecting against Respiratory tract Infections through human Milk Analysis.

We aim to identify mechanisms by which breast feeding prevents respiratory
tract infections. A healthy birth cohort (n=1000) will be set-up and studied
during the first year of life. Human milk will be collected repeatedly and
analysed to provide insights into the protective capacity of human milk
components against (respiratory tract) infections and allergies. Saliva samples
are collected from the infant at 3 and 12 months of age to study the immune
development. Milk samples from a subgroup of 50 extra participants will be used
to specifically investigate the role of extracellular vesicles in the
protective effect of breast milk, using in vitro experiments with a focus on
the development of the immune-liver-brain axis. Additionally, the vesicles will
be characterised and the effect of milk processing will be studied. A subgroup
will be further analyzed to obtain insight in transfer of maternal immunity to
the child. By collecting additional cord blood, amniotic fluid and maternal
blood samples, we will analyze the transfer of maternal immunity. In a
sub-study (N=25), in addition, we will ask participating mothers to complete
four dietary assessment questionnaires, and to collect some saliva and feces of
their infant. With this, we can study the influence of the maternal diet on how
well the breast milk protects their infant against respiratory tract infections
and/or allergies. In the last decades, research shows the importance of the
maternal diet on breast milk composition. However, the performed studies are
often with too small study groups, and studies differ too much in study design
to be able to compare them for meta-analyses. Lastly, there is no sufficient
amount of studies/power to be able to draw conclusions.

Primary objective: 1. To identify the components in human milk (e.g. nutrients,
oligosaccharides, fatty acids and (pathogen specific) immunoglobulins) that
have a protective effect against respiratory tract infections during the first
year of life. Secondary objective: 2. The underlying mechanism of the
components in human milk that offer protection from respiratory tract
infections during the first year of life. 3. To identify the components in
human milk (e.g. nutrients, oligosaccharides, fatty acids and (pathogen
specific) immunoglobulins) that have offer from developing allergies during the
first year of life and to identify the underlying mechanism of these
components. 4. To study the alterations in human milk composition at various
time points. 5. To gain insight in the transfer of maternal immunity to their
child via human milk, amniotic fluid and the placenta.

6.To identify the long-term and short-term influence of the maternal diet on
the breast milk composition.
7. Link the long-term and short-term influence of the maternal diet to the
clinical outcome of respiratory tract infection during the first year of life
8. To derive hypotheses about potential mechanisms if the maternal
diet-influenced components in human milk are protective against respiratory
tract infection during the first year of life.
9. To identify the association between long-term and short-term influence of
the maternal diet on the microbiota composition of a 3-month old infant, and
its consequence for the risk of developing respiratory tract infections in the
first year of life.
10. To identify the antibody repertoire and viral genome in saliva samples from
3 and 12 month-old infants.
11. To link the antibody repertoire in saliva samples with antibody repertoire
in feces and microbiota composition, and how this relates to the risk of
developing respiratory tract infections in the first year of life.
12. To advance our understanding of the effect of human milk extracellular
vesicles on the development of the immune-liver-brain axis, the impact of
maternal factors on human milk extracellular vesicles and the preservation of
their functionality after milk processing.

The study is designed as a prospective observational cohort study, including
1000 healthy mother-child pairs. Directly after birth we will collect data from
all children enrolled through a questionnaire that is send to parents every 2
weeks. The questionnaires will be used to collect data about episodes of
(respiratory tract) infections and already developed allergies during the first
year of age. Meanwhile, we will collect human milk samples at four time points:
within 1 week postpartum and after 1 month, 3 months and 6 months postpartum.
By collecting human milk at these time points, we expect to collect samples
from all relevant time-dependent types of human milk. The human milk samples
will be stored until analysis at the biobank facility of the UMC Utrecht. After
analysis of human milk composition is performed and clinical data collected, we
will compare the two database in order to find beneficial profiles of human
milk components. Additionally we will also collect additional samples to
research what the influence of breastfeeding is on immune development compared
to other routes that are enrolled in the transfer of maternal immunity to the
neonate. This we will de in a subgroup of 20 mother-child dyads. The samples
that we will collect are cord blood samples, a maternal blood sample and an
amniotic fluid sample. Jacobino et al. showed that antibodies retrieved form
human amniotic fluid protected mice pups against RSV-infections (JACOBINO2016).
Very little is known about the protective value antibody titers in cord blood.
Active placental transport of maternal antibodies to the neonatal blood has
been described, but little is known about the effectiveness of these antibodies
(KOHLER1966). To obtain more insight in the effect of the antibodies that are
being transferred by breastfeeding, we will compare the effect of breastfeeding
to the antibodies in cord blood and amniotic fluid. We will also collected
saliva samples in the children enrolled n this subgroup at 1 week, 1, 3, 6, and
12 months postpartum. Saliva samples will be used to study immune development
in children.
Lastly, we will additionally (n=250) ask participating mothers to fill in four
dietary assessment questionnaires, and to collect some saliva and feces from
their infant when he/she is 3 months old.

Only parents will be burdened by participating in this study. Parents will be
visited by researchers four times maximum. Children will not be affected by the
collection of human milk samples, since they are a rather small fraction of the
total amount of breastfeeding. Apart from human milk collection, parents will
have to fill in questionnaires every 2 weeks that will take 1-2 minutes to
complete. Additionally parents will have to fill in three more extensive
questionnaires that will take about 300 minutes each to complete (i.e.
baseline, midterm and end-of-study questionnaires). In total, the maximal
estimated amount of hours will be 5-6 hours for each child. There is a minimal
risk associated with the venipuncture. Although considered safe, rarely
phlebitis, extravasation of blood, bruising and hematoma forming following
venipuncture have been reported. However, since complications form
venipunctures and expressing milk are rare, and we will decrease the risk of a
data breach as best as we can with our DMP, we asses these risks for mothers
acceptable. No risk is associated with the collection of amniotic fluid or
saliva.